Microhyla taraiensis, Khatiwada, Janak Raj, Shu, Guo Cheng, Wang, Shou Hong, Thapa, Arjun, Wang, Bin & Jiang, Jianping, 2017

Khatiwada, Janak Raj, Shu, Guo Cheng, Wang, Shou Hong, Thapa, Arjun, Wang, Bin & Jiang, Jianping, 2017, A new species of the genus Microhyla (Anura: Microhylidae) from Eastern Nepal, Zootaxa 4254 (2), pp. 221-239 : 225-232

Microhyla taraiensis sp. nov.

Holotype: NHM-TU-17A-0110 ( Fig. 3 A, B), an adult male collected from a paddy field in Jamun Khadi , Jhapa district, Nepal, 26.65358°N & 87.91161°E ; elevation 119 m asl; collected by Janak Raj Khatiwada between 19:30 and 20:00 h on 15th May 2015 and deposited in the collection of Tribuvan University , Natural History Museum , Soyambhu, Kathmandu, Nepal .

Suggested common name. Tarai narrow-mouthed frog

Etymology. The species name is derived from the noun “Tarai,” which refers to the flat southern plains of Nepal composed of alluvial soil, where the new species was collected.

Paratypes. NHM-TU-17A-0112 (adult male) and NHM-TU-17A-0111 (adult female) from the same location as the holotype; specimens were deposited in the collection of Tribuvan University , Natural History Museum , Soyambhu, Kathmandu, Nepal.

Diagnosis. The new species is assigned to the genus Microhyla by the following morphological characters: absence of vomerine teeth, hidden tympanum, elliptical tongue, short snout, small eyes not protuberant and invisible when viewed from the ventral side, indistinct canthus rostralis, fingers free of webbing, single outer palmar tubercle, and skin with small tubercles. Microhyla taraiensis sp. nov. is distinguished from other Microhyla species by the following morphological characters: (1) relatively larger body size (SVL = 20.5 mm in male, SVL = 23.7 mm in female); (2) head relatively broad (HL/HW = 0.96–0.83); (3) head length is approximately 79% of head width; (4) short and round snout; (5) hidden tympanum; (6) wider inter-orbital distance approximately 1.5 times greater than the internarial distance; (7) flat and larger inner metacarpal tubercle two times greater than the outer metacarpal; (8) elongated and bean shaped inner metatarsal tubercle; (9) rounded outer metatarsal tubercle (10) toes webbing poorly developed or absent; and (11) light red dots present all over the dorsal surface.

Description of holotype. A medium sized male (SVL = 20.3 mm). Head relatively broad (HW 31.23% of SVL and HL 30% of SVL), snout truncate, eyes not protuberant and not visible when viewed from the ventral side. Canthus rostralis indistinct, nostril closer to the tip of the snout than to the eye (nostril 4.82% and eye 7.19% of SVL), tympanum hidden, supratympanic fold indistinct, moderate eye size (ED 22.80% of HL and ED 8.81% of SVL), pupil round, inter-orbital distance (12.21% of SVL) is greater than the inter-narial distance (8.47% of SVL), elliptical tongue, maxillary and vomerine teeth absent. LA shorter (length of arm 17% of SVL) than LH (length of hand 27% of SVL), fingers thin, free of webbing, finger tips round and not dilated, relative length of fingers from shortest to longest F1 <F2 <F4 <F3. Rounded inner metacarpal tubercle two times greater than outer metacarpal; nuptial pads absent. Subarticular tubercles small, round, formula 1, 2, 3, and 2. Tibiotarsal articulation reached the nostril when the hindlimb is kept parallel to the body. Hindlimbs muscular and slender; length of femur 40.83% of SVL; length of tibia 31.13% of SVL; length of foot 57.33% of SVL. Toes thin and small, toe tips rounded, relative length of toes T1 <T2 <T5 <T3<T4, webbing weakly developed. Elongated and bean-shaped inner metatarsal measuring almost half the length of the first toe. Rounded outer metatarsal tubercle. All five toes had small and round subarticular tubercles, formula 1, 1, 2, 3, and 2.

Skin texture in preservation. Skin on the dorsal and lateral of head and body granular. Skin around the head region smooth, small tubercles present all over the dorsum surface. Small granules present in the cloacal and foot region.

Color in preservative. Skin on the dorsal and lateral surface light brownish-gray; snout gray; arm, femur and tibia brownish-grey; hand and foot (meta-tarsus) creamy white; pupil turned to white from black; iris grey. Small light red spots present all over the dorsal body surface. A rectangular light black mark is present between interorbital spaces; two long light black stripes extend from the orbital region up to the groin. Irregular dark bands are present: one band on the arm, two on the hand, one on the femur and three on the tibia. Belly creamy white; throat light brown in females and light grey in males.

Color in life. Skin light brown with small red spots present all over the dorsal surface of the body, forelimbs and hindlimbs, except on the hand, meta-tarsus and foot. A rectangular black marking was present in the interorbital region; two long black stripes extended from the orbital region to the groin. Irregular dark bands are present: one band on the arm, two on the hand, one on the femur and three on the tibia. Pupil black; iris golden yellow. Belly creamy white; throat brown in the female and blackish gray in males.

Variations and sexual dimorphism. Morphometric variation of type specimens is presented in Table 1. Female body size larger than male body size (t = 4.42, df = 5, p = 0.007). Males had a sub-gular vocal sac, and gravid females contained un-pigmented eggs, which were visible in the belly near the groin.

Morphological comparisons. Based on phylogenetic analysis, Microhyla taraiensis sp. nov. was closely related to M. nilphamariensis and M. ornata . Principal component analysis based on size-corrected values was used to examine the overall morphological variation between Microhyla taraiensis sp. nov. and the M. nilphamariensis population. PCA extracted seven principal component axes with eigenvalues greater than one, and of these, the first two component axes explained 42.47% of the variation ( Table 2 ). The first two principal component axes radially separated the new species from M. nilphamariensis based on hand, finger and toe length ( Figure 4 ). Species with a larger and positive score on PC1 reflected shorter HW, SL, UEW, IOW, IND, LA, LH, F1, F2, F3, F4, FL, LT, LF, T1, T2, T3, T4 and T5, while a negative score signified larger HL, ED and ENL. The second PC with negative scores were associated with species having shorter HL, SL, UEW, ENL, IOW, IND and FL, whereas positive scores were associated with species with larger morphological traits such as HW, ED, LA, LH, F1, F2, F3, F4, LT, LF, T1, T2, T3, T4 and T5.

The SVL of Microhyla taraiensis sp. nov. was significantly larger than that of M. nilphamariensis (t = -5.65, df = 30, P <0.001). In Microhyla taraiensis sp. nov., the head length is approximately 79% of the head width (vs. roughly equal in M. nilphamariensis and M. ornata ), the eye diameter is 35% of the head length (vs. approximately 30% in M. nilphamariensis and 48% in M. ornata ), the upper eyelid is 83% of the eye diameter (vs. 82% in M. nilphamariensis and 39% in M. ornata ), the nostril to eye distance is> 1.5 times that of the nostril to snout distance (vs. <1.5 times in M. nilphamariensis and similar in M. ornata ), and the interorbital distance is <1.5 times that of the internarial distance (vs.>1.5 times in M. nilphamariensis and approximately 3 times in M. ornata ). Microhyla taraiensis sp. nov. has a round inner metacarpal tubercle (vs. oval shape in M. nilphamariensis and goblet-shape in M. ornata ), an elongated outer metacarpal tubercle (vs. small and round in M. nilphamariensis and prominent and heart-shaped in M. ornata ), a round inner metatarsal tubercle (vs. similar in M. nilphamariensis and elongated and very prominent in M. ornata ), and a large and elongated outer metatarsal tubercle (vs. ovoid-shaped, minute, and indistinct in M. nilphamariensis and compressed and large in M. ornata ). Furthermore, a detailed morphometric comparison between the Jamun Khadi population and M. nilphamariensis is presented in Table 3.

TABLE Ɩ. Morphometric measurement of Microhyla taraiensis sp. nov anđ M. nilphamariensis . The results are presenteđ as mean anđ stanđarđ đeviation, with minimum anđ maximum range. All measurements are given in millimeters (mm). Abbreviations are given in Materials anđ Methođs..

Microhyla taraiensis sp.nov. Microhyla nilphamariensis In Microhyla taraiensis sp. nov., the interorbital distance was 0.5 times greater than the internarial distance, while it was <1.3 in M. rubra (Table 3, Howlader et al. 2015). Furthermore, M. rubra has a smaller body size, a shovel-shaped inner metatarsal tubercle, one-third webbed toes, and a black spot on the flanks ( Hasan et al. 2014; Howlader et al. 2015; Wijayathilaka et al. 2016). Likewise, Microhyla taraiensis sp. nov. differs from M. mukhlesuri , M. mymensinghensisn and M. laterite by lacking round expanded discs on the toe tips ( Seshadri et al. 2016; Hasan et al. 2014). Moreover, the new species is morphologically distinct from the other species of the genus Microhyla by lacking toe webbing and toe tips with digital discs (versus presence of these characters in M. achatina , M. annamensis , M. annectens , M. arboricola , M. berdmorei , M. borneensis , M. butleri , M. chakrapanii , M. darevskii , M. fissipes , M. fusca , M. heymonsi , M. karunaratnei , M. laterite , M. maculifera , M. malang , M. mantheyi , M. marmorata , M. mihintalei , M. minuta , M. mixtura , M. nanapollexa , M. okinavensis , M. orientalis , M. palmipes , M. perparva , M. petrigena , M. picta , M. pineticola , M. pulchella , M. pulchra , M. pulverata , M. rubra , M. sholigari , M. superciliaris , and M. zeylanica ) by lacking toe webbing and toe tips with digital discs ( Tschudi 1838; Andersson 1942; Bain & Nguyen 2004; Blyth 1856; Boulenger 1884; Boulenger 1897; Boulenger 1900; Dutta & Ray 2000; Fernando & Siriwardhane 1996; Hallowell 1861; Hu et al. 1966; Inger & Frogner 1979; Inger 1989; Jerdon 1853; Matsui 2011; Parker & Osman-Hill 1948; Parker 1928; Pillai 1977; Poyarkov Jr et al. 2014; Schenkel 1901; Seshadri et al. 2016; Smith 1923; Stejneger 1901; Vogt 1911; Wijayathilaka et al. 2016).

Distribution and habitat. Microhyla taraiensis sp. nov is currently known only from its type locality in Jamun Khadi, Jhapa district, Eastern Nepal (26.65358o N & 87.91161o E; 119 m asl). Jamun Khadi is an artificial wetland with a few scattered Sal trees ( Shorea robusta ). This wetland is surrounded by agricultural lands. Rice is planted twice in a year in the area. During our survey work, most of the areas were fallow and only a few plots were planted with rice. Microhyla taraiensis sp. nov. was collected from fallow land near a rice plantation area. We propose to list this species as Data Deficient (DD) under the IUCN Red List criteria ( IUCN 2001). Most of the males were detected by the loud calls. Microhyla taraiensis sp. nov. is sympatric with Fejervarya sp., Duttaphrynus sp. and Hoplobatrachus sp.

Vocalization. Advertisement calls of Microhyla taraiensis sp. nov. (NHM-TU-17A-0110) were recorded in Jamun Khadi, Nepal on 15th May 2015 between 19:00–23:00. Males start calling at dust and can be heard from approximately 50 m away. The characteristics and structure of a call are shown in Fig. 5 A and 5B. The call sounds were ‘karr…karr…karr….’. Each call consists of several notes, and a total of 10 notes were analyzed from two males. Each note had 13–14 pulses. The intervals between notes and pulses were 0.985±0.106 (1.151 ̶0.818 sec.) and 0.022±0.003 (0.027̶0.015 sec.), respectively. Call duration was 0.75 ± 0.12 (0.688 ̶0.911 sec.). The average dominant frequency was 3305.50 ± 95.46 (3433 ̶3101 Hz). The advertisement call of Microhyla taraiensis sp. nov. was recorded at a water temperature of 25.6°C, a water pH of 4.6, a soil temperature of 28.6°C, a soil moisture of 45%, a soil pH of 6.6, an air temperature of 28.6°C and a relative humidity of 63%.

Range extension of M. nilphamariensis . The molecular analysis revealed that Microhyla species collected from the three populations of central and eastern Nepal formed a single clade in the ML tree. The genetic divergence between Nepalese and type specimens from Nilphamari, Bangladesh is 0.40%. M. nilphamariensis in this study were characterized by the following characters: head length and width almost equal, eye diameter 35% of the head length, upper eyelid 82% of the eye diameter, nostril to eye distance less than one and half times greater than the nostril to snout distance, inter-orbital distance more than one and half times greater than the internarial distance, small oval inner metacarpal, small rounded outer metacarpal tubercle, rounded inner metatarsal tubercle, elongated outer metatarsal tubercle, toes with poorly developed webbing, the absence of digital discs, and irregular back spots present all over the belly ( Fig. 3 D).

TABLE ³. Morphological comparison between Microhyla . Taraiensis sp.nov., M. nilphamariensis anđ M. ornata . Description of M. ornata was đeriveđ from Howlađer et al. (2015b) đue lack of voucher specimens. The results are presenteđ as mean anđ stanđarđ đeviation, with minimum anđ maximum range in parentheses. All measurements are given in millimeters (mm).

Morphological characters Microhyla taraiensis sp.nov. Microhyla nilphamariensis Microhyla ornata

Male (4) Female (³) Male (18) Female (7) Male (6) Female (4) 20.46 2³.7 17.50 19.³7 0 0

:HW 0.90±0.06 0.68±0.17 1.05±0.³1 1.01±0.³³ 0.95±0.0³ 0.97±0.02 (0.9600.8³) (0.8700.5³) (1.6400.61) (1.4600.61) (0.8900.97) (0.9501.00)

:SVL 0.29±0.02 0.18±0.07 0.28±0.080 0.25±0.06 0.28±0.01 0.28±0.02 (0.³000.25) (0.2700.1³) (0.4200.17) (0.³100.15) (0.2700.29) (0.2600.³0)

:HL 0.³2±0.02 0.41±0.02 0.29±0.06 0.³2±0.07 0.49±0.0³ 0.47±0.0³ (0.³400.29) (0.4³00.³7) (0.4100.20) (0.4500.26) (0.4500.54) (0.4³00.50)

UEW:EL 0.67±0.08 1.09±0.4³ 0.87±0.³³ 0.78±0.³1 0.³9±0.0³ 0.³9±0.02 (0.7³00.54) (1.³500.59) (1.5³00.40) (1.2200.41) (0.³400.41) (0.³600.42)

:NS 1.³³±0.17 2.12±0.72 1.29±0.28 1.67±0.8³ 1.51±0.2³ 1.60±0.22 (1.4701.15) (2.5701.29) (1.8500.87) (³.³700.91) (1.2401.92) (1.4³01.92)

:NS 1.52±0.25 1.98±0.³ 6 1.³³ ±0.44 1.6³±0.82 1.68±0.³6 1.77±0.41 (1.7³01.19) (2.2201.57) (2.7800.86) (³.³100.82) (1.³001.86) (1.³702.³2)

:IN 1.4±0.27 1.14±0.1 1.5³±0.³6 1.55±0.³ 6 ³.09 ±0.44 2.87±0.2³ (1.7701.16) (1.2³01.04) (2.2800.72) (1.9701.15) (2.790³.65) (2.60³.12)

: HL 0.42±0.05 0.57±0.08 0.64±0.2³ 0.69±0.09 0.41±0.02 0.³9±0.01 (0.4900.³7) (0.6500.48) (1.1600.³5) (0.800.55) (0.³800.4³) (0.³800.41)

Hand ( Fig. 3 G anđ 3I) ( Fig. 3 K)

metacarpal tubercle rounđ shapeđ smaller anđ oval shapeđ goblet-shapeđ

metacarpal tubercle elongateđ small anđ rounđeđ large, prominent anđ heart-shapeđ

( Fig. 3 H anđ 3J) ( Fig. 3 L)

Advertisement calls of M. nilphamariensis were recorded in Jhuwani, Nepal on 13th May 2015 between 19:00– 23:00. Males start calling after sunset. The characteristics and structure of a call are shown in Fig. 6 A and 6B. The call sounds were ‘karr…karr…karr….’ A total of 10 notes were analyzed from two males. Each note had 19–20 pulses. The interval between notes and pulses were 2.039±0.212 (2.119 ̶1.718 sec.) and 0.016±0.0008 (0. 016– 0.015 sec.), respectively. Each note call duration was 0.357±0.211 (0.506 ̶0.384 sec.). The average dominant frequency was 2595.32±689.85 (3569 2261 Hz). The advertisement call of nilphamariensis was recorded at a water temperature of 25.4°C, a water pH of 4.7, a soil temperature of 27.8°C, a soil moisture of 85%, a soil pH of 6.2, an air temperature of 28.2°C and a relative humidity of 75%. M. nilphamariensis showed a wide range of elevational distribution from 120 to 1690 m in Nepal. All specimens of M. nilphamariensis were observed near rice fields and were sympatric with Fejervarya sp., Duttaphrynus sp., Polypedates sp. and Hoplobatrachus sp.

Figures

FIGURE 3. Morphological illustration of specimens used. A. Dorsal view of male holotype (NHM – TU – 17 A – 0110) of Microhyla taraiensis sp. nov. B. Ventral view of the male holotype C. Dorsal view of M. nilphamariensis collected from Jhuwani, Chitwan, Nepal D. Ventral view of M. nilphamariensis E. Lateral view of head of holotype F. Lateral view of head of M. nilphamariensis G. Ventral view of hand of holotype H. Ventral view of foot of holotype I. Line drawing of hand of holotype showing metacarpal tubercles J. Line drawing of foot of holotype showing metatarsal tubercles K. Ventral view of hand of M. nilphamariensis L. Ventral view of foot of M. nilphamariensis.

FIGURE 4. Plots of the first principal component (PC 1) versus the second (PC 2) of the Microhyla taraiensis sp. nov. versus M. nilphimarensis from a Principal Component Analysis based on size corrected morphometric variables in Appendix S 1.

FIGURE 5. Advertisement call structure of Microhyla taraiensis sp. nov. recorded in Jamun Khadi, Jhapa district, Nepal. A. Oscillogram of a male advertisement call B. Spectrogram showing the dominant frequency.

FIGURE 6. Advertisement call structure of M. nilphamarensis recorded in Jhuwani, Chitwan district, Nepal. A. Oscillogram of a male advertisement call B. Spectrogram showing the dominant frequency.

Tables

TABLE 2. Variable loadings for principal components with Eigen value greater than, from size corrected morphological traits.

Morphological traits	PC 1	PC 2	PC 3	PC 4	PC 5	PC 6	PC 7
HL	-0.05	-0.01	0.81	-0.19	0.01	0.37	-0.04
HW	0.46	0.58	0.10	-0.10	0.18	0.26	0.40
SL	0.12	-0.35	0.65	0.06	-0.35	0.03	-0.43
ED	-0.13	0.10	0.77	-0.19	0.00	0.06	0.14
ENL	-0.24	-0.07	-0.70	0.20	-0.35	0.14	0.23
UEW	0.31	-0.21	-0.67	0.06	0.32	0.25	-0.09
IOW	0.06	-0.11	0.20	-0.10	-0.83	0.01	0.00
IND	0.00	-0.07	-0.03	0.06	0.03	0.02	0.90
LA	0.02	0.16	0.03	-0.13	0.05	0.90	0.02
LH	0.80	0.22	0.26	0.20	-0.10	0.17	-0.09
F1	0.34	0.30	0.29	0.21	0.73	0.01	0.06
F2	0.69	0.13	-0.01	0.21	0.49	-0.02	0.20
F3	0.75	0.10	-0.29	-0.17	0.22	-0.11	0.03
F4	0.60	0.35	-0.28	0.09	0.17	0.13	-0.29
FL	0.24	-0.02	0.07	0.16	-0.06	0.84	0.05
LT	0.62	0.37	-0.07	0.23	-0.20	0.20	0.34
LF	0.59	0.17	-0.02	-0.04	-0.06	0.41	-0.08
T1	0.04	0.20	-0.26	0.89	0.27	0.02	0.01
T2	0.10	0.02	-0.20	0.95	0.03	-0.02	0.05
T3	0.17	0.89	0.03	0.27	0.22	0.01	-0.09
T4	0.35	0.87	0.09	-0.07	0.07	0.12	-0.08
T5	0.11	0.91	0.02	0.07	0.06	0.03	0.04
Eigenvalues	6.12	3.63	2.16	1.69	1.63	1.47	1.27
Cumulative percentage of variance	27.80	44.29	54.09	61.79	69.18	75.87	81.64